The human papillomavirus type 16 E6 and E7 oncoproteins cooperate to induce mitotic defects and genomic instability by uncoupling centrosome duplication from the cell division cycle

Loss of genomic integrity is a defining feature of many human malignancies, including human papillomavirus (HPV)-associated preinvasive and invasive genital squamous lesions. Here we show that aberrant mitotic spindle pole formation caused by abnormal centrosome numbers represents an important mechanism in accounting for numeric chromosomal alterations in HPV-associated carcinogenesis. Similar to what we found in histopathological specimens, HPV-16 E6 and E7 oncoproteins cooperate to induce abnormal centrosome numbers, aberrant mitotic spindle pole formation, and genomic instability. The low-risk HPV-6 E6 and E7 proteins did not induce such abnormalities. Whereas the HPV-16 E6 oncoprotein has no immediate effects on centrosome numbers, HPV-16 E7 rapidly induces abnormal centrosome duplication. Thus our results suggest a model whereby HPV-16 E7 induces centrosome-related mitotic disturbances that are potentiated by HPV-16 E6.

Human Papillomavirus Type 16 E6 and E7 Cause Polyploidy in Human Keratinocytes and Up-Regulation of G2-M-phase Proteins

Human papillomavirus type 16 proteins E6 and E7 have been shown to cause centrosome amplification and lagging chromosomes during mitosis. These abnormalities during mitosis can result in missegregation of the chromosomes, leading to chromosomal instability. Genomic instability is thought to be an essential part of the conversion of a normal cell to a cancer cell. We now show that E6 and E7 together cause polyploidy in primary human keratinocytes soon after these genes are introduced into the cells. Polyploidy seems to result from a spindle checkpoint failure arising from abrogation of the normal functions of p53 and retinoblastoma family members by E6 and E7, respectively. In addition, E6 and E7 cause deregulation of cellular genes such as Plk1, Aurora-A, cdk1, and Nek2, which are known to control the G2-M-phase transition and the ordered progression through mitosis.

Compromised Spindle Assembly Checkpoint due to Altered Expression of Ubch10 and Cdc20 in Human Papillomavirus Type 16 E6 and E7-Expressing Keratinocytes

Cells expressing human papillomavirus type 16 (HPV-16) E6 and E7 proteins exhibit deregulation of G(2)/M genes, allowing bypass of DNA damage arrest signals. Normally, cells with DNA damage that override the G(2) damage checkpoint would precociously enter mitosis and ultimately face mitotic catastrophe and apoptotic cell death. However, E6/E7-expressing cells (E6/E7 cells) have the ability to enter and exit mitosis in the presence of DNA damage and continue with the next round of the cell cycle. Little is known about the mechanism that allows these cells to gain entry into and exit from mitosis. Here, we show that in the presence of DNA damage, E6/E7 cells have elevated levels of cyclin B, which would allow entry into mitosis. Also, as required for exit from mitosis, cyclin B is degraded in these cells, permitting initiation of the next round of DNA synthesis and cell cycle progression. Proteasomal degradation of cyclin B by anaphase-promoting complex/cyclosome (APC/C) is, in part, due to elevated levels of the E2-conjugating enzyme, Ubch10, and the substrate recognition protein, Cdc20, of APC/C. Also, in E6/E7 cells with DNA damage, while Cdc20 is complexed with BubR1, indicating an active checkpoint, it is also present in complexes free of BubR1, presumably allowing APC/C activity and slippage through the checkpoint.

Identification de composé sensibilisant préférentiellement les cellules exprimant les protéines E6 et E7 du VPH à l'irradiation

Le cancer du col utérin (CCU) est dans plus de 99% des cas provoqué par une infection avec le virus du papillome humain (VPH), dont le potentiel oncogénique réside dans l'expression des proto-oncogènes viraux E6/E7. Le potentiel carcinogénique de ces protéines virales réside essentiellement dans leurs actions sur les produits des gènes suppresseurs de tumeur p53 et RB. Les produits de ces gènes, p53 et Rb, font parti des voies de signalisation de réponse aux dommages de l'ADN cellulaire (RDA) et leur perte entraine une perte de fonctionnalité qui mène à une instabilité génomique. À long terme et en présence de d'autres facteurs ceux-ci mèneront au développement d'un cancer. Les protéines E6 et E7 sont constitutivement exprimées dans les cellules du CCU ainsi que dans les cellules de tout autre cancer induit par le VPH et seulement dans ces dernières. La prise en charge des cas avancés de ces cancers se fait principalement par radiothérapie et chimiothérapie concomitante. La chimio-radiothérapie utilisée en traitement est efficace mais résulte en un taux élevé de morbidité et un nombre important de patientes récidiveront. Nous proposons que l'exploitation de l'expression spécifique d’E6 et d’E7 dans les cellules du CCU permette d’envisager une stratégie de létalité synthétique afin d'amplifier l'effet létal de l'irradiation sur les cellules CCU. Ceci permettrait potentiellement d'augmenter l'efficacité du traitement et de diminuer les récidives, ainsi que la morbidité liée au traitement. En s'appuyant sur cette hypothèse, notre objectif est d’identifier des composés dont l'action seule ou couplée à l'irradiation provoquerait préférentiellement la mort des cellules exprimant les protéines E6 et E7 du VPH. Les cellules testées comprennent des cellules isogéniques humaines issues de kératinocytes normaux que nous avons modifiées séquentiellement pour obtenir les modifications associées aux cellules CCU (hTERT, E6 et E7), ainsi que les lignées de cellules de CCU HeLa et CaSki .Nous avons procédé à la mise au point et à la validation du protocole de criblage et des méthodes d’évaluation de la sensibilisation, qui se définit comme une perte de viabilité, un arrêt ou ralentissement de la croissance, par détection d’ATP ainsi que par coloration d’ADN génomique au DRAQ5. Suite à un criblage ciblé impliquant des inhibiteurs connus de la voie de réparation des dommages à l’ADN, nous avons identifié l’inhibiteur de mdm2, Nutlin-3, comme étant un composé sensibilisant et radio-sensibilisant préférentiellement les cellules exprimant E6 et E7 du VPH. La Nutlin-3 a été testée sur des cellules HEKn-hTERT-E6-E7, des cellules CaSki et HeLa. L’effet de sensibilisation et de radio-sensibilisation a été confirmé dans ces trois lignées. Tel que suggéré par son action sur mdmd2, la Nutlin-3 permet la stabilisation de p53 dans les cellules HEKn-hTERT-E6-E7 et CaSki et sa réactivation dans les lignées cellulaires HeLa et CaSki. Malgré cette stabilisation de p53, de façon surprenante, l’effet de la Nutlin-3 sur la sensibilisation et la radio-sensibilisation des cellules HeLa et CaSki semble indépendant de p53, tel qu’observé en utilisant des cellules HeLa-GSE et CaSki-GSE dont le p53 est déficient. In vivo la Nutlin-3a montre dans un essai préliminaire l’inhibition de la croissance tumorale des xénogreffes HeLa chez des souris RAG2γc. Ce résultat reste à confirmer avec un essai impliquant un nombre d’échantillons plus grand. À plus long terme, nous comptons étudier l’implication de mdm2 dans l’effet de sensibilisant de la Nutlin-3 dans les cellules CCUs, ainsi que les autres cibles pouvant être impliquées dans la création de cet effet sensibilisant observé.

Efeito das oncoproteínas virais E6 e E7 do HPV-16 na resposta de células T de pacientes com carcinoma espinocelular de cabeça e pescoço

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Immortalization of distinct human mammary epithelial cell types by human papilloma virus 16 E6 or E7.

Multiple mammary epithelial cell (MEC) types are observed both in mammary ducts in vivo and in primary cultures in vitro; however, the oncogenic potential of different cell types remains unknown. Here, we used human papilloma virus 16 E6 and E7 oncogenes, which target p53 and Rb tumor suppressor proteins, respectively, to immortalize MECs present in early or late passages of human mammary tissue-derived cultures or in milk. One MEC subtype was exclusively immortalized by E6; such cells predominated in late-passage cultures but were rare at early passages and apparently absent in milk. Surprisingly, a second cell type, present only in early-passage tissue-derived cultures, was fully immortalized by E7 alone. A third cell type, observed in tissue-derived cultures and in milk, showed a substantial extension of life span with E7 but eventually senesced. Finally, both E6 and E7 were required to fully immortalize milk-derived MECs and a large proportion of MECs in early-passage tissue-derived cultures, suggesting the presence of another discrete subpopulation. Identification of MECs with distinct susceptibilities to p53- and Rb-targeting human papillomavirus oncogenes raises the possibility that these cells may serve as precursors for different forms of breast cancer.

Detecting uterine cervical cancer cells using molecular biomarkers

Arrière-plan: les cellules tumorales circulantes (CTC) sont détectables dans de nombreux cancers et peuvent être utiles cliniquement pour le pronostic de la maladie, pour mesurer la récidive et pour prédire la sensibilité aux medicaments chimiothérapeutiques. Au cours des dernières années, l’études des CTC dans de nombreux cancers tels que le cancer du sein, du poumon, du côlon et de la prostate a grandement évolué. Alternativement, il y peu d'études à ce sujet concernant le cancer du col de l’utérus (CCU). Objectifs: Notre objectif est d’optimiser le processus d'enrichissement des CTC dans le CCU et la détection moléculaire des biomarqueurs E6 et E7. Matériel et Méthodes: Dans l’optique de mimer la présence de CTC dans le sang, nous avons dilué des cellules cancéreuses CaSki VPH16-positif provenant d’un CCU dans du sang humain prélevé sur des volontaires sains. Les CaSki ont été collectées suite à une centrifugation par densité avec le Ficoll, la lyse des globules rouges (RBC) et la lyse des RBC combinée avec un enrichissement positif et négatif à l’aide de marqueurs de surface cellulaire. Les CTC ont été détectées par la mesure d’expression des oncogènes E6 et E7 du virus du papillome humain (VPH), de la cytokératine 19 (CK19) et de la cycline p16INK4 en utilisant la technique quantitative en temps réel de Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR). Pour valider notre méthode de détection des CTC in vivo, nous avons recruté dix patientes atteintes d’un CCU VPH16 positif et six contrôles sains. Résultats: Dans le modèle de dilutions de cellules CaSki, la lyse des RBC seule ou combinée avec l'enrichissement négatif ou positif suggèrent des limites de détection de 1 CTC par mL de sang pour tous les biomarqueurs moléculaires utilisés. La sensibilité de détection est accrue lors de l'utilisation de l’enrichissement positif et négatif en réduisant le bruit de fond causé par les monocytes sanguins. Contrairement aux oncogènes E6 et E7, les marqueurs CK19 et p16INK4A ont été détectés chez des individus sains, les niveaux d'expression de base appropriés doivent donc être déterminés avec précision par rapport aux patientes CCU. Le gradient de densité par Ficoll a une limite de détection de seulement environ 1000 cellules par mL de sang. Enfin, les CTC ont été détectées dans 2/10 patientes en utilisant le marqueur CK19. Cependant, ces patientes étaient négatives pour les oncogènes E6/E7. Le marqueur p16INK4A était exprimé au même niveau dans tous les échantillons (CCU et normaux). Conclusion: Notre étude suggère que les oncogènes E6 et E7 du VPH16 sont les marqueurs biologiques les plus sensibles et spécifiques en qRT-PCR pour détecter les CTC dans le modèle de dilution de cellules de CCU dans le sang. Chez les patientes atteintes d’un CCU de stade précoce, seulement CK19 a révélé la présence potentielle de CTC, ce qui suggère que ces cellules sont rares à ce stade de la maladie. Mots clés: cancer du col de l’utérus, cellules tumorales circulantes, RT-qPCR, E6 et E7, CK19, p16INK4A, enrichissement immunomagnétique, détection moléculaire.

Evidence for Alteration of EZH2, BMI1, and KDM6A and Epigenetic Reprogramming in Human Papillomavirus Type 16 E6/E7-Expressing Keratinocytes

A number of epigenetic alterations occur in both the virus and host cellular genomes during human papillomavirus (HPV)-associated carcinogenesis, and investigations of such alterations, including changes in chromatin proteins and histone modifications, have the potential to lead to therapeutic epigenetic reversion. We report here that transformed HPV16 E6/E7-expressing primary human foreskin keratinocytes (HFKs) (E6/E7 cells) demonstrate increased expression of the PRC2 methyltransferase EZH2 at both the mRNA and protein levels but do not exhibit the expected increase in trimethylated H3K27 (H3K27me3) compared to normal keratinocytes. In contrast, these cells show a reduction in global H3K27me3 levels in vitro, as well as upregulation of the KDM6A demethylase. We further show for the first time that transformation with the HPV16 E6 and E7 oncogenes also results in an increase in phosphorylated EZH2 serine 21 (P-EZH2-Ser21), mediated by active Akt, and in a downregulation of the PRC1 protein BMI1 in these cells. High-grade squamous cervical intraepithelial lesions also showed a loss of H3K27me3 in the presence of increased expression of EZH2. Correlating with the loss of H3K27me3, E6/E7 cells exhibited derepression of specific EZH2-, KMD6A-, and BMI1-targeted HOX genes. These results suggest that the observed reduction in H3K27me3 may be due to a combination of reduced activities/levels of specific polycomb proteins and increases in demethylases. The dysregulation of multiple chromatin proteins resulting in the loss of global H3K27me3 and the transcriptional reprogramming in HPV16 E6/E7-infected cells could provide an epigenetic signature associated with risk and/or progression of HPV16-associated cancers, as well as the potential for epigenetic reversion in the future.

MicroRNA 203 Expression in Keratinocytes Is Dependent on Regulation of p53 Levels by E6.

A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miRNA 203 (miR-203), which has previously been shown to play an important role in epithelial cell biology by regulating p63 levels. We investigated how expression of human papillomavirus type 16 (HPV16) oncoproteins E6 and E7 affected miR-203 expression during proliferation and differentiation of HFKs. We demonstrated that miR-203 expression is reduced in HFKs where p53 function is compromised, either by the viral oncoprotein E6 or by knockout of p53 using short hairpin RNAs (p53i). We show that the induction of miR-203 observed during calcium-induced differentiation of HFKs is significantly reduced in HFKs expressing E6 and in p53i HFKs. Induction of miR-203 in response to DNA damage is also reduced in the absence of p53. We report that proliferation of HFKs is dependent on the level of miR-203 expression and that overexpression of miR-203 can reduce overproliferation in E6/E7-expressing and p53i HFKs. In summary, these results indicate that expression of miR-203 is dependent on p53, which may explain how expression of HPV16 E6 can disrupt the balance between proliferation and differentiation, as well as the response to DNA damage, in keratinocytes.

miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes

A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7. We demonstrate that the effect on miR-205 is due to E7 activity, as miR-205 expression is dependent on pRb expression. Finally, we provide evidence that miR-24 effects in the cell may be due to targeting of cyclin dependent kinase inhibitor p27. In summary, these results indicate that expression of both miR-24 and miR-205 are impacted by E6 and/or E7 expression, which may be one mechanism by which HPV onco-proteins can disrupt the balance between proliferation and differentiation in keratinocytes.

Immune Responses and Therapeutic Antitumor Effects of an Experimental DNA Vaccine Encoding Human Papillomavirus Type 16 Oncoproteins Genetically Fused to Herpesvirus Glycoprotein D

Recombinant adenovirus or DNA vaccines encoding herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) genetically fused to human papillomavirus type 16 (HPV-16) oncoproteins (E5, E6, and E7) induce antigen-specific CD8(+) T-cell responses and confer preventive resistance to transplantable murine tumor cells (TC-1 cells). In the present report, we characterized some previously uncovered aspects concerning the induction of CD8(+) T-cell responses and the therapeutic anticancer effects achieved in C57BL/6 mice immunized with pgD-E7E6E5 previously challenged with TC-1 cells. Concerning the characterization of the immune responses elicited in mice vaccinated with pgD-E7E6E5, we determined the effect of the CD4(+) T-cell requirement, longevity, and dose-dependent activation on the E7-specific CD8(+) T-cell responses. In addition, we determined the priming/boosting properties of pgD-E7E6E5 when used in combination with a recombinant serotype 68 adenovirus (AdC68) vector encoding the same chimeric antigen. Mice challenged with TC-1 cells and then immunized with three doses of pgD-E7E6E5 elicited CD8(+) T-cell responses, measured by intracellular gamma interferon (IFN-gamma) and CD107a accumulation, to the three HPV-16 oncoproteins and displayed in vivo antigen-specific cytolytic activity, as demonstrated with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled target cells pulsed with oligopeptides corresponding to the H-2D(b)-restricted immunodominant epitopes of the E7, E6, or E5 oncoprotein. Up to 70% of the mice challenged with 5 x 10(5) TC-1 cells and immunized with pgD-E7E6E5 controlled tumor development even after 3 days of tumor cell challenge. In addition, coadministration of pgD-E7E6E5 with DNA vectors encoding pGM-CSF or interleukin-12 (IL-12) enhanced the therapeutic antitumor effects for all mice challenged with TC-1 cells. In conclusion, the present results expand our previous knowledge on the immune modulation properties of the pgD-E7E6E5 vector and demonstrate, for the first time, the strong antitumor effects of the DNA vaccine, raising promising perspectives regarding the development of immunotherapeutic reagents for the control of HPV-16-associated tumors.

Expression of human papillomavirus type 16 E7 oncoprotein alters keratinocytes expression profile in response to tumor necrosis factor-alpha

Acute expression of E7 oncogene from human papillomavirus (HPV) 16 or HPV18 is sufficient to overcome tumor necrosis factor (TNF)-alpha cytostatic effect on primary human keratinocytes. In the present study, we investigated the molecular basis of E7-induced TNF resistance through a comparative analysis of the effect of this cytokine on the proliferation and global gene expression of normal and E7-expressing keratinocytes. Using E7 functional mutants, we show that E7-induced TNF resistance correlates with its ability to mediate pRb degradation and cell transformation. On the other hand, this effect does not depend on E7 sequences required to override DNA damage-induced cell cycle arrest or extend keratinocyte life span. Furthermore, we identified a group of 66 genes whose expression pattern differs between normal and E7-expressing cells upon cytokine treatment. These genes are mainly involved in cell cycle regulation suggesting that their altered expression may contribute to sustained cell proliferation even in the presence of a cytostatic stimulus. Differential expression of TCN1 (transcobalamin I), IFI44 (Interferon-induced protein 44), HMGB2 (high-mobility group box 2) and FUS [Fusion (involved in t(12; 16) in malignant liposarcoma)] among other genes were further confirmed by western-blot and/or real-time polymerase chain reaction. Moreover, FUS upregulation was detected in HPV-positive cervical high-grade squamous intraepithelial lesions when compared with normal cervical tissue. Further evaluation of the role of such genes in TNF resistance and HPVassociated disease development is warranted.

Current trends in the etiology and diagnosis of HPV-related head and neck cancers

Human papilloma virus (HPV) infection is a major risk factor for a distinct subset of head and neck squamous cell carcinoma (HNSCC). The current review summarizes the epidemiology of HNSCC and the disease burden, the infectious cycle of HPV, the roles of viral oncoproteins, E6 and E7, and the downstream cellular events that lead to malignant transformation. Current techniques for the clinical diagnosis of HPV-associated HNSCC will also be discussed, that is, the detection of HPV DNA, RNA, and the HPV surrogate marker, p16 in tumor tissues, as well as HPV-specific antibodies in serum. Such methods do not allow for the early detection of HPV-associated HNSCC and most cases are at an advanced stage upon diagnosis. Novel noninvasive approaches using oral fluid, a clinically relevant biological fluid, allow for the detection of HPV and cellular alterations in infected cells, which may aid in the early detection and HPV-typing of HNSCC tumors. Noninvasive diagnostic methods will enable early detection and intervention, leading to a significant reduction in mortality and morbidity associated with HNSCC.

Functions of human papillomavirus E5 oncogene in epithelial cells and in the onset of cervical cancer

Human papillomaviruses (HPVs) are the causal agents of cervical cancer, which is the second most common cancer among women worldwide. Cellular transformation and carcinogenesis depend on the activities of viral E5, E6 and E7 proteins. Alterations in cell-cell contacts and in communication between epithelial cells take place during cervical carcinogenesis, leading to changes in cell morphology, increased cell motility and finally invasion. The aim of this thesis was to study genome-wide effects of the HPV type 16 (HPV-16) E5 protein on the expression of host cell messenger RNAs (mRNAs) and microRNAs by applying microarray technology. The results showed that the HPV-16 E5 protein alters several cellular pathways involved in cellular adhesion, motility and proliferation as well as in the extracellular matrix. The E5 protein was observed to enhance wound healing of epithelial cell monolayers by increasing cell motility in vivo. HPV-16 E5-induced alterations in the expression of cellular microRNAs and their target genes seem to favour increased proliferation and tumorigenesis. E5 was also shown to affect the expression of adherens junction proteins in HaCaT epithelial keratinocytes. In addition, a study of a membrane cytoskeletal cross-linker protein, ezrin, revealed that when activated, it localizes to adherens junctions. The results suggest that ezrin distribution to forming adherens junctions is due to Rac1 activity in epithelial cells. These studies reveal for the first time the holistic effects of HPV-16 E5 protein in promoting precancerous events in epithelial cells. The results contribute to identifyinging novel markers for cervical precancerous stages and to predicting disease behaviour.